327. THERMOANAEROBACTER SIDEROPHILUS MEDIUM

Tryptone 1.0 g

Peptone (meat) 1.0 g

Yeast extract 1.0 g

K₂HPO₄ 1.6 g

NaH₂PO₄ × 2 H₂O 1.0 g

NH₄Cl 0.5 g

MgSO₄ × 6 H₂O 0.16 g

Trace element solution SL-11 (see below) 1.0 ml

Na-resazurin solution (0.1% w/v) 0.5 ml

CaCl₂ × 2 H₂O 0.06 g

NaHCO₃ 1.0 g

Vitamin solution (see below) 10.0 ml

L-Cysteine-HCl × H₂O 0.3 g

Na₂S × 9 H₂O 0.3 g

Distilled water 1000.0 ml

Trace element solution SL-11:

Na₂-EDTA × 2 H₂O 5.2 g

FeCl₂ × 4 H₂O 1.5 g

ZnCl₂ 70.0 mg

MnCl₂ × 4 H₂O 100.0 mg

H₃BO₃ 6.0 mg

CoCl₂ × 6 H₂O 190.0 mg

CuCl₂ × 2 H₂O 2.0 mg

NiCl₂ × 6 H₂O 24.0 mg

Na₂MoO₄ × 2 H₂O 36.0 mg

Distilled water 1000.0 ml

Dissolve EDTA in 800 ml distilled water, adjust pH to 7 using 2 N NaOH and add ferrous chloride. After ferrous chloride has dissolved add other compounds. Finally adjust pH to 6.0 and bring volume to 1000.0 ml.

Vitamin solution:

Biotin 2.0 mg

Folic acid 2.0 mg

Pyridoxine-HCl 10.0 mg

Thiamine-HCl × 2 H₂O 5.0 mg

Riboflavin 5.0 mg

Nicotinic acid 5.0 mg

D-Ca-pantothenate 5.0 mg

Vitamin B₁₂ 0.1 mg

p-Aminobenzoic acid 5.0 mg

Lipoic acid 5.0 mg

Distilled water 1000.0 ml

Dissolve ingredients (except calcium chloride, bicarbonate, vitamins, cysteine and sulfide), adjust pH to 7.0 and sparge medium with 100% N₂ gas for 30 – 45 min to make it anoxic. Dispense medium under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. After sterilization add calcium chloride, cysteine and sulfide from sterile anoxic stock solutions autoclaved under 100% N₂ gas atmosphere. Add bicarbonate from a sterile anoxic stock solution prepared under 80% N₂ and 20% CO₂ gas mixture. Vitamins are prepared under 100% N₂ gas and sterilized by filtration. The pH of the complete medium should be at 7.0.

 

Updated 01.02.2024